Project Summary/Abstract The most successful cancer immune therapies thus far rely on anticancer T cells to eliminate tumor. If a patient is fortunate enough to possess these antitumor T cells, immune checkpoint inhibitor (CI) therapy can induce a lasting complete response (CR) (Robert et al. 2015). If those T cells are not present, but the patient is fortunate enough to possess a tumor that universally expresses an antigen not also expressed by critical tissues (exemplified by CD19 on ALL), Chimeric Antigen Receptor (CAR) T cells may eliminate the tumor and induce a lasting CR (Sadelain 2015). However, what if a patient does not have antitumor T cells, and has an antigenically diverse, solid tumor that cannot be eliminated by CAR T cells engineered to target one or two antigens? These are the majority of metastatic cancer patients, who continue to succumb to their disease. Pancreatic cancer is one such example of a highly incurable, poorly immunogenic cancer with inhomogenous tumor antigen expression, unresponsive thus far to immune therapies. Antitumor T cells arise through a series of steps. They first require encounter with an Antigen Presenting Cell (APC). The APC must have engulfed a tumor cell, expressed (rather than degraded) the tumor antigens, encountered a reactive T cell, AND expressed costimulatory molecules to activate the T cell (without which, the T cell would have undergone activation-induced cell death). That T cell must then proliferate, maintain activation, and avoid exhaustion. Here, I describe a new therapeutic approach that endows APCs with the genetic responsibility to generate an adaptive antitumor T cell response by achieving all of the above steps. The first component of this approach utilizes a CAR consisting of intracellular phagocytic receptor domains (either TLR4 or FcGRIIA), attached to a tumor-targeting scFv extracellular domain, introduced into APCs. These CAR APCs engulf tumor cells that express the targeted surface antigen (in this case, Lewis A, expressed on 90% of pancreatic cancer cells), and present endogenous tumor antigens. Aim 1 will characterize TLR4 and FcGRIIA CAR APCs? ability to selectively engulf tumor and present endogenous tumor antigens in vitro (1A), and eliminate orthotopic pancreatic cancer in vivo (1B). The second component of this approach utilizes CAR APCs that upon tumor antigen encounter synthesize and express immune modulating molecules that facilitate an adaptive antitumor T cell response. Aim 2 will determine the in vivo function of ?Immune Activating CAR APCs,? which engulf antigen positive (Ag+) tumor cells and subsequently produce a T cell stimulator (41BBL), an APC stimulator (CD40L), or soluble PD1-CTLA4 CIs induced by tumor antigen binding. Ag+ tumor will be injected in the mouse flank, while Ag- tumor will be injected in the pancreas, to separately assess direct CAR APC killing and indirect killing through an adaptive immune response. Finally, I test the additional role of immunogenic and immune sensitizing doses of radiation therapy with CAR APCs to maximize the systemic antitumor response. By developing and characterizing these Immune Activating CAR APCs, I hope to define the steps required to reliably induce an adaptive antitumor T cell response in vivo, and eventually extend a curative immune therapy option to a broader range of cancer patients.